Lineage Tracing Technologies Research Articles

PHD2 inactivation in Type I cells drives HIF-2α-dependent multilineage hyperplasia and the formation of paraganglioma-like carotid bodies.

Key points The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia.Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses.Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia.Inactivation of HIF‐2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia.Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma‐like carotid bodies.These changes, similar to those observed in hypoxia, are dependent on HIF‐2α.Taken together, these findings demonstrate a key role for the PHD2–HIF‐2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body. The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests a rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialized chemoreceptive functions of that organ and, potentially, to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10% oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to (or from) that lineage. Inactivation of a specific HIF isoform, HIF‐2α, in the Type I cells was associated with a greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multilineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF‐2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF‐2α) within Type I cells of the carotid body with respect to the oxygen sensing and adaptive functions of that organ.

Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases.

Endothelial cells and mesenchymal cells are two different cell types with distinct morphologies, phenotypes, functions, and gene profiles. Accumulating evidence, notably from lineage-tracing studies, indicates that the two cell types convert into each other during cardiovascular development and pathogenesis. During heart development, endothelial cells transdifferentiate into mesenchymal cells in the endocardial cushion through endothelial-to-mesenchymal transition (EndoMT), a process that is critical for the formation of cardiac valves. Studies have also reported that EndoMT contributes to the development of various cardiovascular diseases, including myocardial infarction, cardiac fibrosis, valve calcification, endocardial elastofibrosis, atherosclerosis, and pulmonary arterial hypertension. Conversely, cardiac fibroblasts can transdifferentiate into endothelial cells and contribute to neovascularization after cardiac injury. However, progress in genetic lineage tracing has challenged the role of EndoMT, or its reversed programme, in the development of cardiovascular diseases. In this Review, we discuss the caveats of using genetic lineage-tracing technology to investigate cell-lineage conversion; we also reassess the role of EndoMT in cardiovascular development and diseases and elaborate on the molecular signals that orchestrate EndoMT in pathophysiological processes. Understanding the role and mechanisms of EndoMT in diseases will unravel the therapeutic potential of targeting this process and will provide a new paradigm for the development of regenerative medicine to treat cardiovascular diseases.

Endocardial Cell Plasticity in Cardiac Development, Diseases and Regeneration.

Endocardial cells are specialized endothelial cells that form the innermost layer of the heart wall. By virtue of genetic lineage-tracing technology, many of the unexpected roles of endocardium during murine heart development, diseases, and regeneration have been identified recently. In addition to heart valves developed from the well-known endothelial to mesenchymal transition, recent fate-mapping studies using mouse models reveal that multiple cardiac cell lineages are also originated from the endocardium. This review focuses on a variety of different cell types that are recently reported to be endocardium derived during murine heart development, diseases, and regeneration. These multiple cell fates underpin the unprecedented roles of endocardial progenitors in function, pathological progression, and regeneration of the heart. Because emerging studies suggest that developmental mechanisms can be redeployed and recapitulated in promoting heart disease development and also cardiac repair and regeneration, understanding the mechanistic regulation of endocardial plasticity and modulation of their cell fate conversion may uncover new therapeutic potential in facilitating heart regeneration.

Invited commentary

Autogenous vein bypass grafting remains an important therapeutic option for patients with peripheral vascular disease. These vein grafts are durable; however, the development of de novo stenosis within the graft occurs in 30% to 50% of patients within the first several years. Despite multiple therapeutic interventions and vein graft manipulations, the incidence of vein graft disease has not changed over the last 30 years. Basic research has focused on endothelial cells, smooth muscle cells, and extracellular matrix as the responsible culprits for the intimal hyperplastic lesion. However, studies have now shown that the adventitial layer may have a more dynamic role in the intimal hyperplastic lesion. Furthermore, recent studies have demonstrated the potential role of genetic variability in the p27 Kip1 gene as a determinant of clinical outcomes in patients with cardiovascular disease and after clinical interventions. The authors present a provocative in vitro study of human venous adventitial and smooth muscle cells that shows that the p27 single nucleotide polymorphism (p27 KIP1-838C>A) is functional and that the levels of p27 messenger RNA and protein were higher in the cells with the AA genotype compared with the CC genotype. Knockdown of p27 increases growth of both cell types with the AA genotype but not those with the CC genotype. What, however, is groundbreaking in their study is the differential effect of the p27 single nucleotide polymorphism on adventitial cell growth. They noted that adventitial cells of the AA genotype grew significantly more slowly than those with the CC genotype. In this study, only the adventitial cells, and not the smooth muscle cells, are responsible to the inhibitory effects of the protective AA genotype in cell growth. The adventitial layer has been noted to have vascular fibroblasts, perivascular adipocytes, and progenitor cells, which have been shown to affect vascular smooth muscle cell phenotype and growth. How these components interact to achieve the complex intimal hyperplastic lesion is still not known. However, using mouse model technology and lineage tracing technology to discover the cells or cells that are really responsible for the intimal hyperplastic lesion and, therefore, more precisely targeting treatment, could one day allow for the treatment of this disease that has been plaguing long-term vascular surgical outcomes for the last 40 years. The opinions or views expressed in this commentary are those of the authors and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery or the Society for Vascular Surgery. A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cellsJournal of Vascular SurgeryVol. 67Issue 1PreviewCyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. Full-Text PDF Open Archive

Development and maturation of the fibrous components of the arterial roots in the mouse heart.

The arterial roots are important transitional regions of the heart, connecting the intrapericardial components of the aortic and pulmonary trunks with their ventricular outlets. They house the arterial (semilunar) valves and, in the case of the aorta, are the points of coronary arterial attachment. Moreover, because of the semilunar attachments of the valve leaflets, the arterial roots span the anatomic ventriculo‐arterial junction. By virtue of this arrangement, the interleaflet triangles, despite being fibrous, are found on the ventricular aspect of the root and located within the left ventricular cavity. Malformations and diseases of the aortic root are common and serious. Despite the mouse being the animal model of choice for studying cardiac development, few studies have examined the structure of their arterial roots. As a consequence, our understanding of their formation and maturation is incomplete. We set out to clarify the anatomical and histological features of the mouse arterial roots, particularly focusing on their walls and the points of attachment of the valve leaflets. We then sought to determine the embryonic lineage relationships between these tissues, as a forerunner to understanding how they form and mature over time. Using histological stains and immunohistochemistry, we show that the walls of the mouse arterial roots show a gradual transition, with smooth muscle cells (SMC) forming the bulk of wall at the most distal points of attachments of the valve leaflets, while being entirely fibrous at their base. Although the interleaflet triangles lie within the ventricular chambers, we show that they are histologically indistinguishable from the arterial sinus walls until the end of gestation. Differences become apparent after birth, and are only completed by postnatal day 21. Using Cre‐lox‐based lineage tracing technology to label progenitor populations, we show that the SMC and fibrous tissue within the walls of the mature arterial roots share a common origin from the second heart field (SHF) and exclude trans‐differentiation of myocardium as a source for the interleaflet triangle fibrous tissues. Moreover, we show that the attachment points of the leaflets to the walls, like the leaflets themselves, are derived from the outflow cushions, having contributions from both SHF‐derived endothelial cells and neural crest cells. Our data thus show that the arterial roots in the mouse heart are similar to the features described in the human heart. They provide a framework for understanding complex lesions and diseases affecting the aortic root.

Endothelial Cells as Precursors for Osteoblasts in the Metastatic Prostate Cancer Bone

Prostate cancer cells metastasize to the bones, causing ectopic bone formation, which results in fractures and pain. The cellular mechanisms underlying new bone production are unknown. In a recent study, Lin and colleagues, by using state-of-the-art techniques, including prostate cancer mouse models in combination with sophisticated in vivo lineage-tracing technologies, revealed that endothelial cells form osteoblasts induced by prostate cancer metastasis in the bone. Strikingly, genetic deletion of osteorix protein from endothelial cells affected prostate cancer–induced osteogenesis in vivo. Deciphering the osteoblasts origin in the bone microenvironment may result in the development of promising new molecular targets for prostate cancer therapy.

Endothelial cells maintain neural stem cells quiescent in their niche

Niches are specialized microenvironments that regulate stem cells’ activity. The neural stem cell (NSC) niche defines a zone in which NSCs are retained and produce new cells of the nervous system throughout life. Understanding the signaling mechanisms by which the niche controls the NSC fate is crucial for the success of clinical applications. In a recent study, Sato and colleagues, by using state-of-the-art techniques, including sophisticated in vivo lineage-tracing technologies, provide evidence that endothelial amyloid precursor protein (APP) is an important component of the NSC niche. Strikingly, depletion of APP increased NSC proliferation in the subventricular zone, indicating that endothelial cells negatively regulate NSCs’ growth. The emerging knowledge from this research will be important for the treatment of several neurological diseases.

Lung as a Niche for Hematopoietic Progenitors.

Platelets are released from megakaryocytes. The bone marrow has been proposed to be the major site where this process occurs. Lefrançais et al. (2017) using state-of-the-art techniques including two-photon microscopy, in vivo lineage-tracing technologies, and sophisticated lung transplants reveal that the lung is also a primary site for platelet biogenesis. Strikingly, lung megakaryocytes can completely reconstitute platelet counts in the blood in mice with thrombocytopenia. This study also shows that hematopoietic progenitors, with capacity to repopulate the bone marrow after irradiation, are present in the lungs. This work brings a novel unexpected role for the lung as a niche for hematopoiesis. The emerging knowledge from this research may be important for the treatment of several disorders.

Activin Enhances α- to β-Cell Transdifferentiation as a Source For β-Cells In Male FSTL3 Knockout Mice.

Diabetes results from inadequate β-cell number and/or function to control serum glucose concentrations so that replacement of lost β-cells could become a viable therapy for diabetes. In addition to embryonic stem cell sources for new β-cells, evidence for transdifferentiation/reprogramming of non-β-cells to functional β-cells is accumulating. In addition, de-differentiation of β-cells observed in diabetes and their subsequent conversion to α-cells raises the possibility that adult islet cell fate is malleable and controlled by local hormonal and/or environmental cues. We previously demonstrated that inactivation of the activin antagonist, follistatin-like 3 (FSTL3) resulted in β-cell expansion and improved glucose homeostasis in the absence of β-cell proliferation. We recently reported that activin directly suppressed expression of critical α-cell genes while increasing expression of β-cell genes, supporting the hypothesis that activin is one of the local hormones controlling islet cell fate and that increased activin signaling accelerates α- to β-cell transdifferentiation. We tested this hypothesis using Gluc-Cre/yellow fluorescent protein (YFP) α-cell lineage tracing technology combined with FSTL3 knockout (KO) mice to label α-cells with YFP. Flow cytometry was used to quantify unlabeled and labeled α- and β-cells. We found that Ins+/YFP+ cells were significantly increased in FSTL3 KO mice compared with wild type littermates. Labeled Ins+/YFP+ cells increased significantly with age in FSTL3 KO mice but not wild type littermates. Sorting results were substantiated by counting fluorescently labeled cells in pancreatic sections. Activin treatment of isolated islets significantly increased the number of YFP+/Ins+ cells. These results suggest that α- to β-cell transdifferentiation is influenced by activin signaling and may contribute substantially to β-cell mass.

Cytoglobin as a Marker of Hepatic Stellate Cell-derived Myofibroblasts.

Myofibroblasts play important roles in inflammation, fibrosis and tumorigenesis in chronically inflamed liver. Liver myofibroblasts originate from hepatic stellate cells, portal fibroblasts or mesothelial cells, and they are localized in and around fibrotic septum and portal tracts. Liver myofibroblasts are the source of extracellular matrix materials, including type I collagen and multiple fibrogenic growth factors, such as transforming growth factor-β and vascular endothelial growth factor. Although a detailed characterization of the function of individual myofibroblasts has not been conducted, owing to the lack of appropriate cell markers, recent lineage-tracing technology has revealed the limited contribution of myofibroblasts that are derived from portal fibroblasts to various types of liver fibrosis, as compared with the contribution of hepatic stellate cells. In addition, cytoglobin, which is the fourth globin in mammals and function as a local gas sensor, provides a new perspective on the involvement of stellate cells in fibrosis and carcinogenesis, possibly through its anti-oxidative properties and is a promising new marker that discriminates between myofibroblasts derived from stellate cells and those from portal fibroblasts.

Pancreatic Cancer Metastases Harbor Evidence of Polyclonality.

Studies of the cancer genome have demonstrated that tumors are composed of multiple subclones with varied genetic and phenotypic properties. However, little is known about how metastases arise and evolve from these subclones. To understand the cellular dynamics that drive metastasis, we used multicolor lineage-tracing technology in an autochthonous mouse model of pancreatic cancer. Here, we report that precursor lesions exhibit significant clonal heterogeneity but that this diversity decreases during premalignant progression. Furthermore, we present evidence that a significant fraction of metastases are polyclonally seeded by distinct tumor subclones. Finally, we show that clonality during metastatic growth-leading to either monoclonal or polyclonal expansion-differs based on the site of metastatic invasion. These results provide an unprecedented window into the cellular dynamics of tumor evolution and suggest that heterotypic interactions between tumor subpopulations contribute to metastatic progression in native tumors. Studies of tumor heterogeneity indicate that distinct tumor subclones interact during cancer progression. Here, we demonstrate by lineage tracing that metastases often involve seeding by more than one clone and that subsequent cellular outgrowth depends on the metastatic site. These findings provide insight into clonal diversity and evolution in metastatic disease.

Mesenchymal progenitor cells for the osteogenic lineage.

Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.

Location, location, location! The reality of life for an intestinal stem cell in the crypt.

The intestinal crypt has become the archetypal system to understand stem cell behaviour in vivo. Advances in lineage-tracing technology have identified rapidly cycling stem cells at the crypt base with prominent expression of 'marker' genes such as Lgr5. Elegant quantitative analysis of lineage-tracing data has shown that each stem cell within the crypt is in continual neutral competition with the others in order to retain its place in the niche and so prevent differentiation into a specialized lineage. Accordingly, it appears that the regulation of the stem cell pool occurs primarily at the level of the stem cell population, as a simple consequence of competition for the limited space within the niche. However, contradictory data showing that cells located away from the crypt base niche can also sometimes function as stem cells has challenged the notion that stemness is fundamentally cell-extrinsic. Writing in Nature, Ritsma and colleagues have resolved this debate by performing in vivo live-imaging of the crypt base. By tracking individual stem cells over time, they showed that the relative positioning of the cell within the niche stochastically regulates its fate. Stem cells located in close proximity to the crypt base were more likely to persist long-term, but peripheral cells could sometimes move into privileged crypt-base positions. Thus, while many cells within the crypt have stem cell potential, only cells lucky enough to reside in the 'Goldilocks zone' behave as functional stem cells in the long term. The hunt for intestinal stem cells is over: the stem cells are simply found in their niche.

Abstract 4625: An impact of epigenetic approaches targeting JARID1B to modulate malignant behaviors of gastrointestinal cancer.

Abstract Background. Although there are emerging epigenetic factors that contribute to the occurrence, development and metastasis of Gastrointestinal (GI) cancer, the biological roles of epigenetic molecular modifications in different subpopulations, such as cancer stem cells, remains to be understood fully. Here we aimed to clarify the functional roles of the H3K4 demethylase, Jumonji, AT Rich Interactive Domain 1B (JARID1B), an epigenetic factor required for continuous cell growth of melanomas, in GI cancer. Materials and Methods. Based on our ptrevious expression studies showing a high expression level of JARID1B transcript, we focused on four GI cancer cell lines for the analysis: colorectal cancer Colo201 and HCT116; esophageal cancer TE4 and TE8. To study the underlined mechanism, the chromatin immunoprecipitation (ChIP) assay was performed. We performed in vitro experiments, including cell growth, invasion, sphere formation and immunocytochemistry; and in vivo ones, such as chemo-sensitivity and tumorigenesis. For specific knockdown of target genes, lentiviral mediated shRNAs and synthesize siRNA were used. To track biological behaviors of GI cancer cells, a lineage tracing technology was performed to visualize to the mode of tumor cell growth in vitro and in vivo. Result. CD44+/ALDH+ slowly proliferating immature GI cancer populations expressed relatively low levels of JARID1B and the differentiation marker CD20, with a relatively high level of the tumor suppressor p16/INK4A. Fluorescent ubiquitination-based cell cycle indicator transfections to GI cancer cells indicated that endogenous JARID1B increased in the late G1, showing a critical role in G1 to S transition checkpoint. The ChIP assay demonstrated clearly that JARID1B knockdown status leads to the methylation of p16/INK4A gene promoter. Importantly, continuous knockdown of JARID1B resulted in the loss of epithelial differentiation and suppressed cell growth, which associated with phosphorylation induction by Jnk/Sapk, and the irreversible induction of senescence (detected by SA β-galactosidase activity). Finally, green fluorescent-labeled cell tracking indicated that GI cancer cells, which reduced JARID1B expression, had lesser tumorigenicity than JARID1B-positive cells do in vivo. Conclusion. JARID1B plays a critical role in cell cycle regulation at G1/S transition of GI cancer cells. The continuous inhibition of JARID1B would contribute for tumor eradication. Taken together with melanoma study (Cell, 2011), the present study indicated that JARID1B would be a bona fide target for the eradication of chemotherapy-resistant, dormant GI cancer cells, by the induction of endogenous mechanism of cellular senescence. Learning Objective. We identified the novel function and transcriptional targets of epigenetics and verified the important biological behaviors of epigenetics in GI cancer. Citation Format: Katsuya Ohta, Yoshihiro Kano, Masamitsu Konno, Yoshinori Kagawa, Shimpei Nishikawa, Shinichiro Hasegawa, Takahito Fukusumi, Hisataka Ogawa, Atsushi Hamabe, Taroh Satoh, Naotsugu Haraguchi, Hirofumi Yamamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. An impact of epigenetic approaches targeting JARID1B to modulate malignant behaviors of gastrointestinal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4625. doi:10.1158/1538-7445.AM2013-4625

To proliferate or not to proliferate

This editorial refers to ‘Cardiomyocyte cell cycle control and growth estimation in vivo —an analysis based on cardiomyocyte nuclei’ by S. Walsh et al ., pp. 365–373, this issue. Cardiomyocytes in mammalian hearts undergo significant reduction in their cell proliferation capacity soon after birth, which is not disputed. However, it remains uncertain whether the adult heart contains a limited capacity for cell proliferation. Through the use of immunohistochemical staining methods, it has been reported that adult mouse cardiomyocytes can re-enter the cell cycle and duplicate.1,2 However, the origin of the cell-cycle-positive cells was not determined. Others reported permanent inhibition of cell-cycle progression in adult cardiomyocytes in mice.3 With advances in lineage tracing technology, a recent genetic labelling study demonstrated that stem or precursor cells did not contribute significantly to the formation of cardiomyocytes during normal ageing up to 1 year in the mouse. In contrast, precursor cells may participate in the formation of new cardiomyocytes after cardiac injury.4 In humans, high myocyte proliferation rates that could result in the exchange of all cardiomyocytes within 5 years have been reported by employing immunostaining technology on sections of human patients who died of acute myocardial infarction.5 In contrast, using 14C dating analyses in humans, Bergmann et al .6 reported that cardiomyocytes are renewed with a gradual decrease from …

Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes

Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life. Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart.